Golgi Intermediate Compartment Research Articles

Phosphorylation and subcellular localization of transmissible gastroenteritis virus nucleocapsid protein in infected cells

The nucleocapsid (N) protein is the only phosphorylated structural protein of the coronavirus Transmissible gastroenteritis virus (TGEV). The phosphorylation state and intracellular distribution of TGEV N protein in infected cells were characterized by a combination of techniques including: (i) subcellular fractionation and analysis of tryptic peptides by two-dimensional nano-liquid chromatography, coupled to ion-trap mass spectrometry; (ii) tandem mass-spectrometry analysis of N protein resolved by SDS-PAGE; (iii) Western blotting using two specific antisera for phosphoserine-containing motifs; and (iv) confocal microscopy. A total of four N protein-derived phosphopeptides were detected in mitochondria-Golgi-endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-enriched fractions, including N-protein phosphoserines 9, 156, 254 and 256. Confocal microscopy showed that the N protein found in mitochondria-Golgi-ERGIC fractions localized within the Golgi-ERGIC compartments and not with mitochondria. Phosphorylated N protein was also present in purified virions, containing at least phosphoserines 156 and 256. Coronavirus N proteins showed a conserved pattern of secondary structural elements, including six beta-strands and four alpha-helices. Whilst serine 9 was present in a non-conserved domain, serines 156, 254 and 256 were localized close to highly conserved secondary structural elements within the central domain of coronavirus N proteins. Serine 156 was highly conserved, whereas no clear homologous sites were found for serines 254 and 256 for other coronavirus N proteins.

Subcellular distribution of GABAB receptor homo- and hetero-dimers

GBRs (GABA(B) receptors; where GABA stands for gamma-aminobutyric acid) are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. In vitro assays have previously demonstrated that these receptors are heterodimers assembled from two homologous subunits, GBR1 and GBR2, neither of which is capable of producing functional GBR on their own. We have used co-immunoprecipitation in combination with bioluminescence and fluorescence resonance energy transfer approaches in living cells to assess directly the interaction between GBR subunits and determine their subcellular localization. The results show that, in addition to forming heterodimers, GBR1 and GBR2 can associate as stable homodimers. Confocal microscopy indicates that, while GBR1/GBR1 homodimers are retained in the endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartment, both GBR2/GBR2 homodimers and GBR1/GBR2 heterodimers are present at the plasma membrane. Although these observations shed new light on the assembly of GBR complexes, they raise questions about the potential functional roles of GBR1 and GBR2 homodimers.

Capturing protein interactions in the secretory pathway of living cells

The secretory pathway is composed of membrane compartments specialized in protein folding, modification, transport, and sorting. Numerous transient protein-protein interactions guide the transport-competent proteins through the secretory pathway. Here we have adapted the yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) to detect protein-protein interactions in the secretory pathway of living cells. Fragments of YFP were fused to the homooligomeric cargo-receptor lectin endoplasmic reticulum Golgi intermediate compartment (ERGIC)-53, to the ERGIC-53-interacting multi-coagulation factor deficiency protein MCFD2, and to ERGIC-53's cargo glycoprotein cathepsin Z. YFP PCA analysis revealed the oligomerization of ERGIC-53 and its interaction with MCFD2, as well as its lectin-mediated interaction with cathepsin Z. Mutation of the lectin domain of ERGIC-53 selectively decreased YFP complementation with cathepsin Z. Using YFP PCA, we discovered a carbohydrate-mediated interaction between ERGIC-53 and cathepsin C. We conclude that YFP PCA can detect weak and transient protein interactions in the secretory pathway and hence is a powerful approach to study luminal processes involved in protein secretion. The study extends the application of PCA to carbohydrate-mediated protein-protein interactions of low affinity.

Molecular analysis of a novel hereditary C3 deficiency with systemic lupus erythematosus

A case of inherited homozygous complement C3 deficiency (C3D) in a patient with systemic lupus erythematosus (SLE) and the molecular basis for this deficiency are reported. A 22-year-old Japanese male was diagnosed as having SLE and his medical history revealed recurrent tonsillitis and pneumonia. He was diagnosed as having C3D because of undetectable serum C3 level. His parents were consanguineous. Sequence analysis of C3D cDNA revealed a homozygous deletion of exon 39 (84 bp). A single base substitution (AG to GG) in the 3′-splice acceptor site of intron 38 was identified by sequencing the genomic DNA. Expression of C3Δ(ex39) cDNA, the C3cDNA lacking exon 39, in COS-7 cells revealed that C3Δ(ex39) was retained in endoplasmic reticulum–Golgi intermediate compartment because of defective secretion. These data indicate that a novel AG → GG 3′-splice acceptor site mutation in intron 38 caused aberrant splicing of exon 39, resulting in defective secretion of C3.

A Golgi-Localized ERK

Extracellular signal-regulated kinases (ERKs) are members of the mitogen-activated protein kinase (MAPK) family best known for being activated by phosphorylation in response to various stimuli, after which these proteins can accumulate in the nucleus and regulate gene expression by phosphorylation of nuclear targets. Bind et al. revisited the ERK3 member of this family, which has been reported to have undergone substantial divergence between the rat and the mouse and human homologs in terms of protein size and localization. Resequencing of the rat gene revealed a missing G. The protein predicted by this revised sequence is essentially identical to the mouse and human forms, and the size of the predicted protein was confirmed by in vitro translation and immunoblot analysis with an antibody against the C-terminal region. The previously reported form was truncated and did not include this C-terminal region. More interestingly, the authors used this C-terminally directed antibody to show localization of ERK3 to the Golgi and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). When transfected cells expressing a green fluorescent protein (GFP)- and FLAG-tagged version of ERK3 (GFP-FL-ERK3) were analyzed, the protein was detected in the Golgi and ERGIC compartments and in the nucleus with the C-terminal antibody. In contrast, an antibody that recognizes a region within the N-terminal catalytic domain only labeled GFP-FL-ERK3 in the nucleus. Furthermore, truncation of the C terminus produced a form that was only detected in the speckled nuclear pattern, suggesting that a motif in the C terminus may be responsible for the Golgi and ERGIC localization and that cleavage of the C terminus may be a mechanism for regulation of ERK3 localization. Indeed, ERK3 has the sequence KHLN in its C-terminal domain, which is predicted to be an ER retention and retrieval motif, and mutation of the lysine resulted in an increase in the proportion of cells exhibiting a completely nuclear localization. The abundance of ERK3 in the nucleus varied with the cell cycle, based on the analysis of synchronized cultured cells, such that ERK3 was shifted from the Golgi and ERGIC compartments to the nucleus after G1. After completion of mitosis, ERK3 was predominantly localized to the Golgi and ERGIC. In the presence of proteasome inhibitors, a fragment under 80 kD was detected with the C-terminal antibody, consistent with the possibility that C-terminal cleavage contributes to ERK3 regulation. Deletion of the region containing two potential caspase-like cleavage motifs in the C-terminal region of ERK3 resulted in loss of cleavage products and a decrease in the proportion of cells in which ERK3 was localized to the nucleus. Thus, ERK3 may be retained in internal membranes and cleaved through a cell cycle-regulated mechanism to release a truncated form that can translocate to the nucleus. E. Bind, Y. Kleyner, D. Skowronska-Krawczyk, E. Bien, B. D. Dynlacht, I. Sánchez, A novel mechanism for mitogen-activated protein kinase localization. Mol. Biol. Cell 15 , 4457-4466 (2004). [Abstract] [Full Text]

A novel mechanism for mitogen-activated protein kinase localization.

Mitogen-activated protein kinases/extracellular signal regulated kinases (MAPKs/ERKs) are typically thought to be soluble cytoplasmic enzymes that translocate to the nucleus subsequent to their phosphorylation by their activating kinases or mitogen-activated protein/extracellular signal regulated kinase kinase. We report here the first example of nuclear translocation of a MAPK that occurs via temporally regulated exit from a membranous organelle. Confocal microscopy examining the subcellular localization of ERK3 in several cell lines indicated that this enzyme was targeted to the Golgi/endoplasmic reticulum Golgi intermediate compartment. Deletion analysis of green fluorescent protein (GFP)-ERK3 uncovered a nuclear form that was carboxy-terminally truncated and established a Golgi targeting motif at the carboxy terminus. Immunoblot analysis of cells treated with the proteasome inhibitor MG132 further revealed two cleavage products, suggesting that in vivo, carboxy-terminal cleavage of the full-length protein controls its subcellular localization. In support of this hypothesis, we found that deletion of a small region rich in acidic residues within the carboxy terminus eliminated both the cleavage and nuclear translocation of GFP-ERK3. Finally, cell cycle synchronization studies revealed that the subcellular localization of ERK3 is temporally regulated. These data suggest a novel mechanism for the localization of an MAPK family member, ERK3, in which cell cycle-regulated, site-specific proteolysis generates the nuclear form of the protein.

VAMP-associated protein-A regulates partitioning of oxysterol-binding protein-related protein-9 between the endoplasmic reticulum and Golgi apparatus

We recently showed that oxysterol-binding protein (OSBP), one of twelve related PH domain containing proteins with lipid and sterol binding activity, interacts with VAMP-associated protein (VAP)-A on the endoplasmic reticulum (ER). In addition to OSBP, seven OSBP-related proteins (ORPs) bind VAP-A via a conserved E-F/Y-F/Y-DA ‘FFAT’ motif. We focused on this interaction for ORP9, which is expressed as a full-length (ORP9L) or truncated version missing the PH domain (ORP9S). Mutation analysis showed that the interaction required the ORP9 FFAT motif and the N-terminal conserved domain of VAP. Endogenous ORP9L displayed Golgi localization, which was partially mediated by the PH domain based on limited localization of OPR9–PH–GFP with the Golgi apparatus. When inducibly overexpressed, ORP9S and ORP9L colocalized with VAP-A and caused vacuolation of the ER as well as retention of the ER–Golgi intermediate compartment marker ERGIC-53/p58 in the ER. ORP9L mutated in the VAP-A binding domain (ORP9L-FY→AA) did not localize to the ER but appeared with giantin and Sec31 on large vesicular structures, suggesting the presence of a hybrid Golgi–COPII compartment. Normal Golgi localization was also observed for ORP9L-FY→AA. Results show that VAP binding and PH domains target ORP9 to the ER and a Golgi–COPII compartment, respectively, and that ORP9L overexpression in these compartments severely perturbed their organization.

Localization of a highly active pool of type II phosphatidylinositol 4-kinase in a p97/valosin-containing-protein-rich fraction of the endoplasmic reticulum.

Different phosphoinositides are synthesized in cell membranes in order to perform a variety of functions. One of the most abundant of these lipids is phosphatidylinositol (PI) 4-phosphate (PI4P), which is formed in human eukaryotes by type II and type III phosphatidylinositol 4-kinase (PI4K II and III) activities. PI4K II activity occurs in many different subcellular membranes, although no detailed analysis of the distribution of this activity has been reported. Using density gradient ultracentrifugation, we have previously found that in A431 cells the predominant PI4K activity arises from a type II alpha enzyme that is localized to a buoyant membrane fraction of unknown origin [Waugh, Lawson, Tan and Hsuan (1998) J. Biol. Chem. 273, 17115-17121]. We show here that these buoyant membranes contain an activated form of PI4K II alpha that can be separated from the bulk of the PI4K II alpha protein in A431 and COS-7 cells. Proteomic analysis revealed that the buoyant membrane fraction contains numerous endoplasmic reticulum (ER)-marker proteins, although it was separated from the bulk of the ER, ER-Golgi intermediate compartment, transitional ER, Golgi and other major subcellular membranes. Furthermore, the majority of the cytoplasmic valosin-containing protein (VCP), an AAA+ATPase implicated in various ER, transitional ER, Golgi and nuclear functions, was almost completely localized to the same buoyant membrane fraction. Co-localization of VCP and PI4K activity was confirmed by co-immunoprecipitation. These results suggest the previously unsuspected existence of an ER-related domain in which the bulk of the cellular PI4P synthesis and VCP are localized.

Mammalian Erv46 localizes to the endoplasmic reticulum-Golgi intermediate compartment and to cis-Golgi cisternae.

Yeast endoplasmic reticulum (ER) vesicle protein Erv46p is a novel membrane protein involved in transport through the early secretory pathway. Investigation of mammalian Erv46 (mErv46) reveals that it is broadly expressed in tissues and protein-secreting cells. By immunofluorescence microscopy, mErv46 displays a crescent-shaped perinuclear staining pattern that is characteristic of the Golgi complex. Quantitative immunoelectron microscopy indicates that mErv46 is restricted to the cis face of the Golgi apparatus and to vesicular tubular structures between the transitional ER and cis-Golgi. Minor amounts of mErv46 reside in ER membranes and later Golgi cisternae. On Brefeldin A treatment, mErv46 redistributes to punctate structures that costain for ERGIC53. Depletion of mErv46 protein by RNA interference caused no apparent structural changes in the intermediate compartment or Golgi complex. These findings place mErv46 in a group of itinerant proteins that cycle between the ER and Golgi compartments such as ERGIC53 and the p24 proteins.

Direct interaction of the Golgi membrane with the endoplasmic reticulum membrane caused by nordihydroguaiaretic acid

Nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, blocks protein transport from the endoplasmic reticulum (ER) to the Golgi complex and induces the redistribution of Golgi proteins into the ER. We investigated characteristics of NDGA-induced retrograde movement of the Golgi proteins to the ER. At an early stage of incubation of cells with NDGA, the Golgi complex formed convoluted membrane aggregates. Electron microscopy revealed that these aggregates directly interact en bloc with the ER membrane. The direct interaction and subsequent incorporation of the Golgi proteins into the ER were found to be temperature-dependent. The protein of ER–Golgi intermediate compartment (ERGIC), ERGIC53, was rapidly accumulated in the Golgi upon treatment with NDGA. This accumulation was significantly inhibited by low temperature at 15 °C. Under the condition, the redistribution of the Golgi proteins into the ER as well as the direct interaction between the ER and the Golgi by NDGA were also inhibited, suggesting an important role of the ERGIC in the retrograde movement. In contrast, the low temperature did not inhibit formation of the Golgi aggregates by NDGA. Taken together, these results suggest that NDGA causes the redistribution of the Golgi proteins into the ER through the direct connections between the Golgi, the ERGIC, and the ER.

Dolichos biflorus agglutinin-binding site expression in basal keratinocytes is associated with cell differentiation

A basal layer of squamous epithelia such as epidermis contains stem cells, transit amplifying cells as well as postmitotic differentiating cells. A detailed knowledge of the transition among these cell types in the course of epidermal renewal is important. It would help in better understanding of many pathological processes, including cancer, and in employment of epidermal cells for therapeutic purposes. In this study we analyzed the possible role of Dolichos biflorus agglutinin (DBA)-reactive α-N-acetylgalactosamine glycosylation in behavior of the human epidermal basal cells under in vivo and in vitro conditions. The data received from porcine epidermis were also included. Part of basal cells was positive for DBA-binding sites and these cells exhibited a lower presence of β 1 integrin in their basal surface connected to the basement membrane. The perinuclear Golgi-like accumulation of β 1 integrin was observed in some cultured keratinocytes. The co-localization of integrin with DBA-binding sites and 58 kDa protein suggests that α-N-acetylgalactosamine glycosylation could be related to β 1 integrin retention in the endoplasmatic reticulum Golgi intermediate compartment (ERGIC) at the beginning of the secretory pathway. The lack of anchorage in culture elevated the number of DBA-binding site positive cells without significant influence on cell growth when cells isolated directly from epidermis were employed in study. Some role of DBA-reactive glycoligand expressions in a suprabasal movement of differentiated basal cells can be hypothesized.

Lectins and protein traffic early in the secretory pathway.

Lectins of the early secretory pathway are involved in selective transport of newly synthesized glycoproteins from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC). The most prominent cycling lectin is the mannose-binding type I membrane protein ERGIC-53 (ERGIC protein of 53 kDa), a marker for the ERGIC, which functions as a cargo receptor to facilitate export of an increasing number of glycoproteins with different characteristics from the ER. Two ERGIC-53-related proteins, VIP36 (vesicular integral membrane protein 36) and a novel ERGIC-53-like protein, ERGL, are also found in the early secretory pathway. ERGL may act as a regulator of ERGIC-53. Studies of ERGIC-53 continue to provide new insights into the organization and dynamics of the early secretory pathway. Analysis of the cycling of ERGIC-53 uncovered a complex interplay of trafficking signals and revealed novel cytoplasmic ER-export motifs that interact with COP-II coat proteins. These motifs are common to type I and polytopic membrane proteins including presenilin 1 and presenilin 2. The results support the notion that protein export from the ER is selective.

Expression of caveolin by bovine lymphocytes and antigen-presenting cells.

Caveolin is a generic term for a family of proteins that include caveolin-1, -2 and -3. Although the distribution of these proteins varies between cells, caveolin-1 and -2 are commonly found coating membrane invaginations known as caveolae. Studies on human and murine cells suggest that caveolin/caveolae can be found in neutrophils, macrophages and mast cells, in which they are involved in the uptake of pathogens, but not in lymphocyte cell lines. Expression of caveolin-1, -2 and -3 in bovine immune cells was investigated using confocal microscopy and Western blotting. Staining for caveolin-1 was evident in all peripheral blood mononuclear cells (PBMC) and in CD4+, CD8+ and CD21+ lymphocytes, monocytes, macrophages and monocyte-derived dendritic cells (DC). In addition, the caveolin-1 antibody detected a protein with a molecular weight of approximately 22[?]000 in all PBMC, macrophages and DC, as well as in bovine aortic endothelial (BAE)-1 cells and human endothelial cells by Western blotting. In macrophages and DC, caveolin co-localized with the endoplasmic reticulum--Golgi intermediate compartment (ERGIC) and to a lesser extent with Golgi, but not with endoplasmic reticulum. Staining was not seen on the plasma membrane in any bovine immune cells, suggesting the absence of caveolae, while in BAE-1 cells staining was predominantly on the cell membrane. Caveolin-2 could not be detected in any bovine cells by confocal microscopy or Western blotting, while caveolin-3 was detected in all bovine cells by Western blotting, but not by confocal microscopy. These data provide evidence for the presence of caveolin in bovine lymphocytes and antigen-presenting cells.

The Glycoprotein gp48 of Murine Cytomegalovirus: PROTEASOME-DEPENDENT CYTOSOLIC DISLOCATION AND DEGRADATION

Degradation of misfolded or unassembled proteins that are co-translationally inserted into the endoplasmic reticulum involves the cytosolic proteasome system. Different principles may exist for the export of proteins into the cytosol for proteasomal degradation. Here we studied the degradation pathway of the viral glycoprotein gp48, a type I transmembrane protein, encoded by the m06 gene of murine cytomegalovirus. In cells stably transfected with the cytomegalovirus m06 gene or infected with the virus itself, two populations of gp48 can be distinguished that have different fates. Complexes of gp48 and the major histocompatibility complex (MHC) class I molecule, are transported to the lysosome for degradation. Unassembled gp48 is degraded by the cytosolic proteasome. Proteasomal inhibitors stabilize the unassembled gp48 in its core-glycosylated and membrane-associated form in the endoplasmic reticulum (ER)-Golgi intermediate compartment. This implicates that both endoplasmic reticulum and ER-Golgi intermediate compartment export gp48 and that degradation is coupled to a functional proteasome. Analysis of gp48 mutants revealed that the cytosolic part of gp48 was not responsible for the proteasome-dependent substrate transport out of the ER-Golgi intermediate compartment. Thus an indirect interaction between the proteasome and its substrate has to be discussed.

Structural evidence for multiple transport mechanisms through the Golgi in the pancreatic beta-cell line, HIT-T15.

Accurate data on the three-dimensional architecture of the Golgi is prerequisite for evaluating the mechanisms of transit through this organelle. Here we detail the structure of the Golgi ribbon within part of an insulin-secreting cell in three dimensions at approximately 6 nm resolution. Rapid freezing, freeze-substitution and electron tomography were employed. The Golgi in this region is composed of seven cisternae. The cis-most element is structurally intermediate between the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and the cis-most cisterna characterized in three dimensions at high resolution in a normal rat kidney cell [Ladinsky, Mastronarde, McIntosh, Howell and Staehelin (1999) J. Cell Biol. 144, 1135-1149]. There are three trans-cisternae that demonstrate morphological and functional variation. The membrane surface areas and volumes of these elements decrease from cis to trans. The two trans-most cisternae are dissociated from the stack and are fragmented by tubulation. ER closely adheres to and inserts between individual trans-cisternae. Many of the 2119 small, clathrin-negative vesicles that are in close proximity to the Golgi fill the region where trans-cisternae have moved out of register with the ribbon. These data provide evidence that cisternal progression/maturation, trafficking via membrane tubules and vesicle-mediated transport act in concert in the same region of the Golgi ribbon, and suggest an important role for the ER in regulating membrane dynamics at the trans-Golgi.

Infectious bronchitis virus envelope protein targeting: implications for virus assembly.

Coronaviruses acquire their membrane envelope by budding into the lumen of Golgi and pre-Golgi compartments. After budding, virions are thought to move in vesicles through the secretory pathway, and exit the cell when these vesicles fuse with the plasma membrane (Holmes et al., 1981, Tooze et al., 1987). The specific compartment into which coronaviruses bud is the cis-Golgi network (CGN), also known as the endoplasmic reticulum Golgi intermediate compartment (Krijnse Locker et al., 1994). Just as enveloped viruses that bud from the plasma membrane must direct the accumulation of their envelope proteins at the cell surface, coronaviruses must localize their envelope proteins to the membranes of the CGN.

Golgi apparatus immunolocalization of endomannosidase suggests post-endoplasmic reticulum glucose trimming: implications for quality control.

Trimming of N-linked oligosaccharides by endoplasmic reticulum (ER) glucosidase II is implicated in quality control of protein folding. An alternate glucosidase II-independent deglucosylation pathway exists, in which endo-alpha-mannosidase cleaves internally the glucose-substituted mannose residue of oligosaccharides. By immunogold labeling, we detected most endomannosidase in cis/medial Golgi cisternae (83.8% of immunogold labeling) and less in the intermediate compartment (15.1%), but none in the trans-Golgi apparatus and ER, including its transitional elements. This dual localization became more pronounced under 15 degrees C conditions indicative of two endomannosidase locations. Under experimental conditions when the intermediate compartment marker p58 was retained in peripheral sites, endomannosidase was redistributed to the Golgi apparatus. Double immunogold labeling established a mutually exclusive distribution of endomannosidase and glucosidase II, whereas calreticulin was observed in endomannosidase-reactive sites (17.3% in intermediate compartment, 5.7% in Golgi apparatus) in addition to the ER (77%). Our results demonstrate that glucose trimming of N-linked oligosaccharides is not limited to the ER and that protein deglucosylation by endomannosidase in the Golgi apparatus and intermediate compartment additionally ensures that processing to mature oligosaccharides can continue. Thus, endomannosidase localization suggests that a quality control of N-glycosylation exists in the Golgi apparatus.

Potential role for protein kinases in regulation of bidirectional endoplasmic reticulum-to-Golgi transport revealed by protein kinase inhibitor H89.

Recent evidence suggests a regulatory connection between cell volume, endoplasmic reticulum (ER) export, and stimulated Golgi-to-ER transport. To investigate the potential role of protein kinases we tested a panel of protein kinase inhibitors for their effect on these steps. One inhibitor, H89, an isoquinolinesulfonamide that is commonly used as a selective protein kinase A inhibitor, blocked both ER export and hypo-osmotic-, brefeldin A-, or nocodazole-induced Golgi-to-ER transport. In contrast, H89 did not block the constitutive ER Golgi-intermediate compartment (ERGIC)-to-ER and Golgi-to-ER traffic that underlies redistribution of ERGIC and Golgi proteins into the ER after ER export arrest. Surprisingly, other protein kinase A inhibitors, KT5720 and H8, as well as a set of protein kinase C inhibitors, had no effect on these transport processes. To test whether H89 might act at the level of either the coatomer protein (COP)I or the COPII coat protein complex we examined the localization of betaCOP and Sec13 in H89-treated cells. H89 treatment led to a rapid loss of Sec13-labeled ER export sites but betaCOP localization to the Golgi was unaffected. To further investigate the effect of H89 on COPII we developed a COPII recruitment assay with permeabilized cells and found that H89 potently inhibited binding of exogenous Sec13 to ER export sites. This block occurred in the presence of guanosine-5'-O-(3-thio)triphosphate, suggesting that Sec13 recruitment is inhibited at a step independent of the activation of the GTPase Sar1. These results identify a requirement for an H89-sensitive factor(s), potentially a novel protein kinase, in recruitment of COPII to ER export sites, as well as in stimulated but not constitutive Golgi-to-ER transport.

A Visual Intracellular Classification Strategy for Uncharacterized Human Proteins

The human cDNA and genomic sequencing projects will result in the identification and isolation of some 140,000 genes, the majority of which lack predicted functions and for which the cellular localizations are not known. The identification and characterization of protein components of specific cell structures and machineries are essential steps not only toward defining functions of genes but also toward understanding cell function and regulation. We describe here a new approach, termed PROLOC, which uses full-length cDNAs for systematic classification of novel proteins as a functional pointer. We have PCR-amplified 25 uncharacterized human genes and expressed the encoded proteins as GFP fusions in a human cell line. This pilot project has identified novel proteins associated with the nucleolus, mitochondria, the ER, the ER–Golgi-intermediate compartment (ERGIC), the GC, the plasma membrane, and cytoplasmic foci. This visual classification approach may be scaled up to handle a large number of novel genes and permit the generation of a global cellular protein localization map. Such information should be valuable for many aspects of functional genomics and cell biology.

The neuronal ceroid lipofuscinosis CLN8 membrane protein is a resident of the endoplasmic reticulum

Progressive epilepsy with mental retardation (EPMR) is a new member of the neuronal ceroid lipofuscinoses (NCLs). The CLN8 gene underlying EPMR was recently identified. It encodes a novel 286 amino acid transmembrane protein that contains an endoplasmic reticulum (ER)-retrieval signal (KKRP) in its C-terminus. A homozygous mutation in the orthologous mouse gene (Cln8) underlies the phenotype of a naturally occurring NCL model, the motor neuron degeneration mouse (mnd). To characterize the product of the CLN8 gene and to determine its intracellular localization, we expressed CLN8 cDNA in BHK, HeLa and CHO cell lines. In western blotting and pulse-chase analyses an approximately 33 kDa protein that does not undergo proteolytic processing steps was detected. Using CLN8 and cell organelle specific antibodies with confocal immunofluorescence microscopy the CLN8 protein was shown to localize in the ER. Partial localization to the ER-Golgi intermediate compartment (ERGIC) was also observed. The ER-ERGIC localization was not altered in the CLN8 protein representing the EPMR mutation. However, mnd mutant protein was only found in the ER. Mutations in the ER retrieval signal KKRP resulted in localization of CLN8 to the Golgi apparatus. Taken together, these data strongly suggest that CLN8 is an ER resident protein that recycles between ER and ERGIC.